2,2'-[ethylenebis(oxyphenyl-2,1-eneazo)]bis[N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxobutyramide

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-08-15 to 2011-09-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to relevant guidelines and compliant to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

Deviations:

yes

Remarks:

: Instead of the test system generated of a mixed inoculum from different sources, inoculum of the aqueous phase of non-adapted activated sludge was used.

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemikaliengesetzt and directive 88/320/EEC

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Inoculum of the aqueous phase of non adapted activated sludge. Municipal sewage treatment plant, D-31137 Hildesheim. Activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.
- Pretreatment: The activated sludge was washed twice with autoclaved tap water. After washing, the supernatant was decanted and the settled sludge was maintained in an aerobic condition by aeration with CO2 free air until test start.
- Inoculation: 10 ml/L inoculum (functional control: 4 ml/L) corresponding to the final concentration of 100 mg/L (functional control: 40 mg/L) suspended solids was used to initiate inoculation.
- Initial cell/biomass concentration: 10 mL/L inoculum was used to initiate inoculation
- Water filtered: no

Duration of test (contact time):

28 d

Initial conc.:

30 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: Mineral salts medium acc. to OECD 302C
- Test temperature: 25.0 - 25.5 °C
- pH:
pH-Values in the Test Vessels at Test Start and Test End
pH-Value
Start End
Inoculum Control - 1) 6,42
2) 6,24
Functional Control - 1) 6,40
2) 6,58
Test Item 7,28 1) 6,40
2) 6,36
3) 6,36
Blank - 8,23

- pH adjusted: no
- Aeration of dilution water: Continuous stirring
- Continuous darkness: yes


TEST SYSTEM
- Culturing apparatus: Brown glass bottles, volume 500 mL
- Number of culture flasks/concentration: Triplicates
- Method used to create aerobic conditions: Continuous stirring
- Measuring equipment: The oxygen consumption was determined in the incubation vessels by the OxiTop measuring system at 360 measuring points during the 28 d incubation period.
- Details of trap for CO2 and volatile organics if used: A rubber sleeve with soda lime was hung into the opening of the bottles. The bottles were closed with OxiTop measuring heads and the measuring system was activated.


SAMPLING
- Sampling frequency: The temperature in the incubator was documented continuously throughout the test with a hygrothermograph.
The colony forming units and concentration of suspended solids of the inoculum were determined at test start.
The pH of the test item solution was determined in a separate replicate at test start. No adjustment of pH was necessary.
The pH of the test solutions was determined at test end.
The oxygen consumption was determined in the incubation vessels by the OxiTop measuring system at 360 measuring points during the 28 d incubation period.


CONTROL AND BLANK SYSTEM
- Inoculum blank: 30 mg/L test item in aqua demin.
- Abiotic sterile control: No
- Functional control: Anilin, 25 mg/L
- Inoculum control: Test medium and inoculum without test and reference item.

Reference substance:

aniline

Test performance:

Based on the calculated oxygen demand (ThOD), the test concentration of 30 mg/L corresponding to an oxygen demand of
38.1 mgO2/L in the vessel was selected.

The test solutions were prepared in measuring flasks and given into brown glass bottles as incubation vessels (inoculum control, functional control) or were prepared directly in brown glass
bottles (test item, blank):
• 3 incubation vessels for the test item (P1, P2, P3)
• 2 incubation vessels for the functional control (R1, R2)
• 1 incubation vessel for the blank (B1)
• 2 incubation vessels for the inoculum control (C1, C2)

A separate test item replicate was prepared for pH measurement at test start:
• 1 incubation vessel for the test item (PpH).

The test item was weighed out and was transferred directly into the brown glass bottles with 250 mL test medium (consisting of the appropriate volumes of mineral medium stock solutions, de-mineralised water and inoculum) for the test item replicates and with 250 mL demineralised water for the blank, respectively.

For the functional control the reference item was weighed out and was transferred into a 500 mL measuring flask with demineralised water. The appropriate volumes of mineral medium stock solutions and inoculum were added, then the flask was filled up with demineralised water.

The inoculum control, consisting of mineral medium stock solutions, demineralised water and inoculum, was prepared in a 500 mL measuring flask.
The test solutions for reference item and inoculum control were divided using 250 mL measuring flasks before being filled into the brown glass bottles.
A rubber sleeve with soda lime was hung into the opening of the bottles. The bottles were closed with OxiTop measuring heads and the measuring system was activated.

Parameter:

% degradation (O2 consumption)

Value:

10

Sampling time:

28 d

Details on results:

Colony forming units (CFU) of the inoculum were determined at test start by standard dilution plate count: 2.0 x 109 CFU/L.
The concentration of suspended solids of the inoculum at test start was 9.79 g/L corresponding to 97.9 mg/L in the test vessels of the test item and control replicates and 39.2 mg/L in the vessels of the reference item replicates.

Results with reference substance:

The functional control reached the pass level of > 65 % after 4 days in the 1st replicate and after 10 days in the 2nd replicate. It came to a biodegradation rate of 100 % after 28 days. Hence the validity criterion that the degradation should be > 65 % after 14 days was fulfilled.

Biological Oxygen Demand (BOD) of Inoculum Control, Functional
Control and Blank

		BOD [mg O2/L]
		Inoculum Control	Functional Control 25 mg/L				Blank 30 mg/L
			R1		R2		B1
Date	[d]	mean^*	gross	Net	gross	net	gross
16.08.2011	1	2.2	0.0	-2.2	0.0	-2.2	-5.6
17.08.2011	2	4.8	0.0	-4.8	0.0	-4.8	-1.4
18.08.2011	3	7.3	40.9	33.6	11.3	4.0	-4.2
19.08.2011	4	9.3	49.3	40.0	39.5	30.2	0.0
20.08.2011	5	11.8	59.2	47.4	42.3	30.5	0.0
21.08.2011	6	13.2	63.4	50.2	45.1	31.9	0.0
22.08.2011	7	14.9	69.0	54.1	46.5	31.6	0.0
23.08.2011	8	16.1	74.7	58.6	47.9	31.8	0.0
24.08.2011	9	16.6	77.5	60.9	50.7	34.1	1.4
25.08.2011	10	17.5	80.3	62.8	53.5	36.0	0.0
26.08.2011	11	18.0	83.1	65.1	56.4	38.4	-1.4
27.08.2011	12	18.3	84.5	66.2	56.4	38.1	-2.8
28.08.2011	13	19.4	87.4	68.0	60.6	41.2	-1.4
29.08.2011	14	20.3	90.2	69.9	63.4	43.1	0.0
30.08.2011	15	21.1	91.6	70.5	66.2	45.1	-1.4
31.08.2011	16	21.4	91.6	70.2	66.2	44.8	-1.4
01.09.2011	17	23.1	94.4	71.3	69.0	45.9	1.4
02.09.2011	18	24.0	94.4	70.4	69.0	45.0	0.0
03.09.2011	19	24.5	95.8	71.3	69.0	44.5	1.4
04.09.2011	20	26.2	97.2	71.0	70.5	44.3	2.8
05.09.2011	21	26.8	98.6	71.8	70.5	43.7	2.8
06.09.2011	22	28.2	98.6	70.4	73.3	45.1	1.4
07.09.2011	23	28.2	98.6	70.4	71.9	43.7	1.4
08.09.2011	24	29.3	98.6	69.3	77.5	48.2	0.0
09.09.2011	25	29.3	100.0	70.7	77.5	48.2	1.4
10.09.2011	26	30.4	101.0	70.6	87.4	57.0	-1.4
11.09.2011	27	31.3	103.0	71.7	90.2	58.9	2.8
12.09.2011	28	31.6	104.0	72.4	91.6	60.0	2.8

   *) corrected values due to different inoculum concentration in inoculum control and functional control

Biological Oxygen Demand (BOD) of Test Item

			BOD [mg O2/L]
		Inoculum Control	Test Item 30 mg/L
			P1		P2		P3
Date	[d]	mean	gross	net	gross	net	gross	net
16.08.2011	1	5.6	7.0	1.4	5.6	0.0	4.2	-1.4
17.08.2011	2	12.0	16.9	4.9	15.5	3.5	15.5	3.5
18.08.2011	3	18.3	18.3	0.0	16.9	-1.4	16.9	-1.4
19.08.2011	4	23.3	28.2	5.0	28.2	5.0	26.8	3.6
20.08.2011	5	29.6	33.8	4.2	33.8	4.2	32.4	2.8
21.08.2011	6	33.1	38.0	4.9	38.0	4.9	36.6	3.5
22.08.2011	7	37.3	42.3	5.0	42.3	5.0	40.9	3.6
23.08.2011	8	40.2	45.1	4.9	45.1	4.9	43.7	3.5
24.08.2011	9	41.6	49.3	7.7	49.3	7.7	46.5	4.9
25.08.2011	10	43.7	49.3	5.6	49.3	5.6	46.5	2.8
26.08.2011	11	45.1	49.3	4.2	50.7	5.6	47.9	2.8
27.08.2011	12	45.8	46.5	0.7	47.9	2.1	46.5	0.7
28.08.2011	13	48.6	50.7	2.1	52.1	3.5	50.7	2.1
29.08.2011	14	50.7	52.1	1.4	53.5	2.8	52.1	1.4
30.08.2011	15	52.9	53.5	0.6	55.0	2.2	53.5	0.6
31.08.2011	16	53.6	53.5	0.0	56.4	2.9	55.0	1.5
01.09.2011	17	57.8	59.2	1.4	62.0	4.2	60.6	2.8
02.09.2011	18	59.9	60.6	0.7	63.4	3.5	63.4	3.5
03.09.2011	19	61.3	63.4	2.1	67.6	6.3	67.6	6.3
04.09.2011	20	65.6	64.8	-0.8	70.5	5.0	70.5	5.0
05.09.2011	21	67.0	66.2	-0.8	73.3	6.3	73.3	6.3
06.09.2011	22	70.5	66.2	-4.2	74.7	4.3	74.7	4.3
07.09.2011	23	70.5	67.6	-2.8	76.1	5.7	76.1	5.7
08.09.2011	24	73.3	67.6	-5.7	77.5	4.3	78.9	5.7
09.09.2011	25	73.3	69.0	-4.3	80.3	7.1	80.3	7.1
10.09.2011	26	76.1	67.6	-8.5	78.9	2.9	78.9	2.9
11.09.2011	27	78.2	73.3	-4.9	84.5	6.3	84.5	6.3
12.09.2011	28	78.9	74.7	-4.2	84.5	5.6	84.5	5.6

Validity criteria fulfilled:

yes

Remarks:

The percentage degradation of the functional control reached the pass level > 65 % after 14 days. The difference of extremes of replicate values of biodegradation of the test item at the end of the test was less than 20 %.

Interpretation of results:

not inherently biodegradable

Conclusions:

Under our test conditions, the test item was not inherently biodegradable within 28 days.

Executive summary:

The inherent biodegradability of the test item was determined in the Modified MITI Test (II) with non adapted activated sludge for a period of 28 days. The study was conducted from 2011-08-15 to 2011-09-12 according to OECD Guideline 302 C. The test item concentration selected as appropriate was 30 mg/L, corresponding to a ThOD of 38.1 mgO₂/L. The oxygen was depleted by respiration of bacteria and the degradation was followed by measuring the oxygen concentration. The biodegradation is therefore expressed as the percentage BOD depletion and was calculated for each study day.

The mean oxygen depletion in the inoculum control was 78.9 mg O₂/L on day 28.

In order to check the activity of the test system aniline was used as functional control. The functional control reached the pass level of > 65 % after 4 days in the 1^st replicate and after 10 days in the 2^nd replicate. It came to a biodegradation rate of 100 % after 28 days.

The 1^st test item replicate reached the 10 % level (beginning of biodegradation) after 2 days and came to a maximum of 20 % after 9 days. The 2^ndtest item replicate reached the 10 % level after 4 days and came to a maximum of 20 % after 9 days. The biodegradation of the 3^rd test item replicate reached the 10 % level after 9 days and reached 15 % on day 28. The pass level > 70 % for evidence of inherent, ultimate biodegradability was not reached within 28 days. After 28 days the mean biodegradation rate was 10 %.

The mean oxygen depletion in the abiotic control was 2.8 mg O₂/L on day 28.

The validity criteria of the guideline are fulfilled.

Under our test conditions, the test item was not inherently biodegradable within 28 days.

Biodegradation of the Test Item in Comparison to the
                   Functional Control

		Biodegradation [%]
		Study Day [d]
	Replicate	7	14	21	28
Test Item 30 mg/L	1	13	4	0	0
	2	13	7	17	15
	3	9	4	17	15
	mv	12	5	11	10
Functional Control 25 mg/L	1	90	100	100	100
	2	52	72	73	100
	mv	71	86	87	100

                            mv = mean value

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Justification for type of information:

see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Acetolone Pigments – Nanoforms and Bulk Forms (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Key result

Parameter:

% degradation (O2 consumption)

Value:

10

Sampling time:

28 d

Validity criteria fulfilled:

yes

Interpretation of results:

not inherently biodegradable

Conclusions:

Under our test conditions, the test item was not inherently biodegradable within 28 days.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Description of key information

According to the criteria set in Annex XI of REGULATION (EC) No 1907/2006 a ready biodegradation study is scientifically unnecessary based on the following arguments applied for the Acetolone Pigments. Since it is generally known that pigments are not or only marginally (bio)degradable, the Persistance-criterion is accepted for Acetolone pigments.

Therefore only one biodegradation study was performed. As Acetolone Pigments are of very poor water solubility, rather inherent than ready biodegradability was assessed for Pigment Yellow 154 in the Modified MITI Test (II) according to OECD 302 C. The substance was not inherently biodegradable.